Nodulation Experiment by Cross-Inoculation of Nitrogen-Fixing Bacteria Isolated from Root Nodules of Several Leguminous Plants

Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes. This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes. Forty-six isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes. Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively. The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes. The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA). Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation. Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max. The gene expression studies revealed that GM5 had a higher expression of the nifH gene. The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen. These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max). This suggested the potential application of these rhizobia as biofertilizer. Further studies are required to verify the N2-fixing efficiency of the isolates.


Introduction
The global food production supply is under increasingly severe stress due to the expanding human population and adverse environmental conditions.While synthetic nitrogen fertilizers have addressed those problems by providing a solution for efficient crop production and contributing to the growing world's food supply, the excessive application of chemical fertilizer is one of the leading causes of pollution of groundwater.Polluted groundwater is an increasing hazard to the health of both humans and the environment [1].Environmentally friendly alternative fertilizers are crucial for the sustainability of agriculture.Biological nitrogen fixation (BNF) offers a sustainable and cost-effective alternative to chemical fertilizer for use on legumes.BNF improves the soil fertility by fixing the atmospheric nitrogen (N 2 ) into biologically available ammonium, which the plants then utilize to synthesize various biomolecules.This process is performed exclusively by prokaryotes, including archaea and bacteria [2].BNF accounts for 65% of the nitrogen utilized for the effective production of crops, which demonstrates the economic importance of rhizobia in agriculture [3].
The 'nod' strategy is a mechanism to induce root nodule formation in legumes [4,5].Legumes have an exceptional ability to develop a symbiotic relationship with rhizobia.The symbiotic association between legumes and rhizobia is highly specific, and each rhizobial species interacts only with particular legumes and vice versa.Rhizobia such as Rhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Allorhizobium, and Sinorhizobium establish symbiotic associations by chemotactically responding to flavonoid molecules which are released by the host legumes as signals.These flavonoid compounds induce the expression of nodulation genes (nod) in the Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes.This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes.Fortysix isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes.Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively.The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes.The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA).Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation.Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max.The gene expression studies revealed that GM5 had a higher expression of the nifH gene.The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen.These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max).This suggested the potential application of these rhizobia as biofertilizer.Further studies are required to verify the N 2 -fixing efficiency of the isolates.
94 o C for 30 s, annealing at 58 o C for 1 min, and extension at 72 o C for 30 s, with a final extension at 72 o C for 7 min.The amplification of the nodC gene was performed using the specific primer set of NodC_F 540 and NodC_R [25]; with initial denaturation at 94 o C for 3 min, followed by 30 cycles of denaturation at 94 o C for 45 s, annealing at 50 o C for 30 s, and extension at 72 o C for 1 min, and a final extension at 72 o C for 5 min.Agarose gel electrophoresis (1.5%) was used to visualize the amplified PCR product.The PCR products were purified using an AccuPrep PCR/Gel Purification Kit, and were sequenced on an ABI3730 using BigDye Terminator v3.1 (Thermo Fisher Scientific, USA).

Symbiotic Properties
The three types of soybean seeds used in the plant experiment were: Trifolium repens, Glycine max, and Phaseolus vulgaris.All of the seeds used were surface-sterilized with a 2% sodium hypochlorite solution (2 min) and 70% ethanol (1 min), and then rinsed several times with sterile distilled water.The seeds were germinated in 1% water-agar media under dark conditions at 28°C for 2-3 days, and the treated seeds were then planted in pots filled with vermiculite (with one seed per pot).Two samples were used as the controls: one was inoculated with Shewanella oneidensis MR-1 [28], and the other was not inoculated with any microbial strains.All of the selected strains and controls were grown in an R2A liquid medium and inoculated in the soil, with as much as 3×10 8 cells, using a syringe.The plants were grown for 40 days under controlled conditions in the incubator at 28°C, with 16 h for light and 8 h for dark conditions.The plants were harvested 40 days after seeding and were checked for whether root nodules had formed.They were then dried in an oven at 70°C.The dry weight of the roots and root nodules were recorded.All of the experiments were conducted in triplicate.

Indole Acetic Acid (IAA) Production
The Salkowski method was used in order to determine whether the isolated strain had produced an indole compound [29].All of the strains used in the experiment were cultured in an R2A liquid medium containing Ltryptophan (0.5 g/l), and were then incubated for seven days at 28°C at 90 rpm.The two control samples were used as negative controls: one was inoculated with S. oneidensis MR-1, and the other control was not inoculated with any bacterium.After 7 days, the culture solution was centrifuged at 10,000 rpm for 1 min to collect the supernatant and filtered using a 0.45 μl filter with the syringe.One ml of the supernatant was mixed with 2 ml of Salkowski reagent (which was dissolved in 4.5 g of iron chloride in 1 L of 10.8 M sulfuric acid), and was then incubated at room temperature for 30 min.A color change to pink indicated the production of indole.IAA production was quantified from the supernatant absorbance using a spectrophotometer with a wavelength of 530 nm.The experiment was conducted in triplicates, and the average was used to calculate the final concentration (μM) by substituting it into the calibration curve.The relative fold increase in IAA production was calculated based on the control value.

Quantitative Reverse Transcription PCR (qRT-PCR) Analysis of nifH Gene
The expression levels of the nifH gene were determined by quantitative reverse transcription PCR (qRT-PCT) analysis.Bacterial RNAs were extracted from the root nodules of the Glycine max that was inoculated with the strains of GM5 and WC24, using and RNeasy Plant Mini Kit (Qiagen, Germany).The liquid nitrogen-frozen nodules were crushed in the homogenizer to extract the RNA.A first strand cDNA synthesis was carried out by the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio Inc., Japan) following the manufacturer's instructions.The qRT-PCR was performed on a CFX Connect Real-Time System (Bio-Rad Laboratories, Inc., USA) using the AccuPower GreenStar RT-qPCR Premix (Bioneer), and primer sets of Pol [24] and IGK3+DVV [10] for GM5 and WC24 strains, respectively.The primer sequences are given in Table 2.The expression levels of the nifH were determined and compared to the housekeeping gene GAPDH [30].

Results
Isolation and Amplification of the Housekeeping (16S rRNA) and Symbiotic (nifH and nodC) Genes In this study, 46 strains were isolated from plant root nodules collected from Trifolium repens (WC), Wisteria floribunda (WF), Pisum sativum (PS), Vigna radiate (ND), and Glycine max (GM).High-quality genomic DNA was procured from each isolate and amplified using gene-specific primers for the 16S rRNA, nifH, and nodC genes.Among the 46 isolated strains, only four isolates -three from Trifolium repens (WC15, WC16, and WC24) and one from Glycine max (GM5) -were used.They were selected for their morphologies on agar plates and for the successfully acquired 16S rRNA gene sequences.The rest of the isolates showed morphological similarities or were unsuccessful in their sequencing of the 16S rRNA gene.The nifH gene was amplified using the IGK3/DVV and PolF/PolR primer pairs, and an amplicon of the desired length (300~400 bp) was produced.Only one isolate (GM5) produced the desired 600 bp fragment from the nodC gene using the nodC540F/1160R primer pair.The amplified products of the 16S rRNA, nifH, and nodC genes were sequenced and compared using the NCBI BLAST tool.Based on 16S rRNA gene sequences, four out of 46 tested isolates -WC15, WC16, WC24, and GM5 -were identified as Rhizobium sp., Sphingomonas sp., Methylobacterium sp., and Bradyrhizobium sp., respectively.The top three BLASTn scores of each gene sequence are presented in Table 3.

Detection of the Indole Acetic Acid (IAA) Production in Bacterial Isolates
The studied isolates -Bradyrhizobium sp.1.97 fold) when compared to the control group (42.6 ± 10.12 μM).This confirmed that the selected strains produced IAA (Fig. 4).S. oneidensis MR-  1 was used as a negative control in the present study because it lacked a homolog to the TnpA (tryptophan-indol lyase) enzyme that converts tryptophan to indol, which resulted in a low level of IAA (12.7 ± 8.01 μM) in the MR-1 strain.The MR-1 utilizes tryptophan-based signaling molecules in Biofilm preparation [28].A statistical analysis using a one-way ANOVA test revealed a significant difference between the control and the treated samples (*p value < 0.05).

Symbiotic Roperties of the Isolated Bacterial Strain
The surface-sterilized seeds of Trifolium repens, Glycine max, and Phaseolus vulgaris were germinated on a 1% water-agar media for 2-3 days, transferred to the pots at one seed per pot, and allowed to grow for approximately 40 days under controlled conditions.On the third day, the bacterial isolates WC15 (from T. repens), WC16 (from T. repens), WC24 (from T. repens), GM5 (from G. max) were inoculated into each pot.The formation of pink nodules was observed on the roots of Glycine max inoculated with WC24 and GM5, which suggested that these strains were effective potentially in nitrogen fixation in Glycine max.In contrast, root nodules were not observed in the experimental pots of T. repens and P. vulgaris, inoculated with the isolated strains of WC15, WC16, WC24, and GM5, suggesting that they were not microsymbionts for these particular legumes.These finding indicated that the Methylobacterium strain (WC24), isolated from the root nodules of T. repens was able to induce nodulation in G. max.The effect of the nodulation on the dry weight of the root was evaluated.The plant roots inoculated with WC24 weighted the most (0.038 ± 0.017 g), followed by GM5 (0.035 ± 0.002 g).As shown in Table 4, the number of nodules and dry nodule weight (g) per plant was higher in the plants inoculated with GM5 than those inoculated with the WC24.The cross-section of the root nodules collected from the G. max that were inoculated with the isolated bacteria (GM5 and WC24) indicated a potential symbiotic hemoglobin (leghemoglobin), observed by a red color (Fig. 5).

Absolute Expression of the nifH Gene Using qRT-PCR
The nifH gene is commonly used as a marker gene to track N 2 fixation in plants.The expression of the nifH gene in both strains (WC24 and GM) that showed nodulation was quantified using qRT-PCR.The GM5 (Bradyrhizobium sp.) showed a higher expression of the nifH gene, measured at 6.27 × 10 10 ± 1.39 × 10 10 gene copy number per 1 g of G. max root nodules.The WC24 (Methylobacterium sp.) produced 4.97 × 10 10 ± 2.53 × 10 9 gene copy number per 1 g of G. max root nodules (Fig. 6).A higher expression of the nifH gene is usually related to a higher N 2 fixation ability of plants.The existence and expression of the nifH gene in both isolates indicated the N 2 fixation ability of the G. max, with both the host plant-originated strain GM5 and the non-host plant-originated strain WC24.

Discussion
The N 2 -fixing ability of rhizobia is essential for adding nitrogen to the soil, which in turn increases the soil fertility and crop productivity.The association of rhizobia and legumes is highly specific, and each rhizobial strain establishes a symbiotic relationship with a specific legume plant.The host specificity of rhizobia is observed at both the genus and species levels [6].A certain level of mismatch between the two symbiotic partners is tolerated for the development of symbiosis.The 16S rRNA gene sequencing and phylogeny were successfully used to identify the rhizobial symbionts collected from the root nodules of five different leguminous plants (Trifolium repens, Wisteria floribunda, Pisum sativum, Vigna radiate, and Glycine max).The tested rhizobia -WC15, WC16, WC24, and GM5 -displayed the highest percentage of similarity to the genera Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively.Previous studies have reported the potential of the fulllength 16S rRNA gene sequence to accurately identify bacterial species with a high taxonomic resolution [27,[31][32][33][34].
The phylogenetic relationships among the bacterial isolates were tested based on their symbiotic genes (nifH and nodC).The neighbor-joining phylogenies of the nifH and nodC genes were consistent with the 16S rRNA gene-based phylogeny, which suggested that these symbiotic genes were coevolved [27,32,[35][36].Incongruencies in the nifH and nodC phylogenies have been previously reported [37,38], and a higher sequence similarity of the nodC gene was reported in Bradyrhizobium and Mesorhizobium spp.[39].This supported the nodulation of G. max in this study, which was inoculated by a non-host plant-originated bacterium.The present study demonstrated that most of the bacterial isolates were able to produce significant amounts of IAA in the presence of tryptophan.In in vivo conditions, the isolated rhizobia utilized L-Tryptophan as a substrate for the synthesis of IAA (auxin), which controls the various physiological processes in plants [18,19,21,40].Previous reports have confirmed that IAA is produced by different symbiotic and non-symbiotic nitrogen-fixing bacteria [20,41,42].
A cross inoculation experiment evaluated the nodulation and N 2 fixation ability in relation to the isolated strains: Rhizobium (WC15), Sphingomonas (WC16), Methylobacterium (WC24), and Bradyrhizobium (GM5).These were used as inoculants for the three different leguminous plant species, T. repens, G. max, and P. vulgaris.The finding showed that the Methylobacterium strain (WC24) from T. repens nodules induced nodulation in the G. max roots, and produced a significant number of nodules.Root nodules were observed in the G. max infected with Bradyrhizobium (GM5).The Bradyrhizobium species -particularly B. elkanii, B. japonicum, B. diazoefficiens, B. liaoningense, and B. yuanmingense -are the native nodulating species of G. max, whereas Methylobacterium is not a native nodulating bacterium for G. max [43,44].The cross-section of the root nodules collected from the infected G. max indicated the presence of leghemoglobin (hemeprotein).Leghemoglobin is a symbiotic hemoglobin protein that creates the anaerobic conditions inside the nodules which is necessary for the effective symbiotic nitrogen fixation [11,12,45].
A previous report showed that the expression of the nifH gene was positively correlated with a higher nitrogen fixation [24,46].The expression of the nifH gene in the isolated strains of WC24 and GM5 indicated their high N fixation ability in the G. max plant.This study's findings suggested that there was a symbiotic compatibility between G. max and the Methylobacterium strain from T. repens nodules.The nodulation capacity and nitrogenfixing ability of the isolated bacteria indicated the possibility of using these eco-friendly microbial agents as biofertilizers.While these findings are a beginning to developing a biofertilizer, the nitrogen fixation efficiency of the infected G. max needs to be evaluated under field conditions and a greater number of isolated strains still need to be evaluated with other leguminous plants to truly develop an effective universal biofertilizer.

Fig. 1 .
Fig. 1.Phylogenetic tree of nitrogen-fixing bacteria (WC15, WC16, WC24, and GM5) isolated from leguminous root nodules based on 16S rRNA gene sequence.The number indicates the levels of bootstrap support based on 10,000 replicates.

Fig. 3 .
Fig. 3. Phylogenetic tree of nodC gene from a nitrogen-fixing bacterium (GM5).The number indicates the levels of bootstrap support based on 10,000 replicates.